1. Field of the Invention
This invention relates to oligonucleotides that are complementary to mammalian insulin-like growth factor II (IGF II) genes which oligonucleotides modulate tumor cell growth in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.
2. References
The following publications, patent applications and patents are cited in this application:
1. Toretsky, J. A. and Helman, L. J. Involvement of IGF-II in human cancer, J Endocrinol. 149: 367-72, 1996.
2. Werner, H. and LeRoith, D. The role of the insulin-like growth factor system in human cancer, Adv Cancer Res. 68: 183-223, 1996.
3. Rogler, C. E., Yang, D., Rossetti, L., Donohoe, J., Alt, E., Chang, C. J., Rosenfeld, R., Neely, K., and Hintz, R. Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice, J Biol Chem. 269: 13779-84, 1994.
4. Bates, P., Fisher, R., Ward, A., Richardson, L., Hill, D. J., and Graham, C. F. Mammary cancer in transgenic mice expressing insulin-like growth factor II (GF-II) [see comments], Br J Cancer. 72: 1189-93, 1995.
5. Cullen, K. J., Lippman, M. E., Chow, D., Hill, S., Rosen, N., and Zwiebel, J. A. Insulin-like growth factor-II overexpression in MCF-7 cells induces phenotypic changes associated with malignant progression, Mol Endocrinol. 6: 91-100, 1992.
6. Werner, H., Adamo, M., Roberts, C. T., Jr., and LeRoith, D. Molecular and cellular aspects of insulin-like growth factor action, Vitam Horm. 48: 1-58, 1994.
7. Curcio, L. D., Bouffard, D. Y., and Scanlon, K. J. Oligonucleotides as modulators of cancer gene expression, Pharmacol Ther. 74: 317-32, 1997.
8. Narayanan, R. and Akhtar, S. Antisense therapy, Curr Opin Oncol. 8: 509-15, 1996.
9. Ho, P. T. and Parkinson, D. R. Antisense oligonucleotides as therapeutics for malignant diseases, Semin Oncol. 24: 187-202, 1997.
10. Crooke, S. T. and Bennett, C. F. Progress in antisense oligonucleotide therapeutics, Annu Rev Pharmacol Toxicol. 36: 107-29, 1996.
11. Christofori, G., Naik, P., and Hanahan, D. A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis, Nature. 369: 414-8, 1994.
12. El-Badry, O. M., Minniti, C., Kohn, E. C., Houghton, P. J., Daughaday, W. H., and Helman, L. J. Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors, Cell Growth Differ. 1: 325-31, 1990.
13. Kim, K. W., Bae, S. K., Lee, O. H., Bae, M. H., Lee, M. J., and Park, B. C. Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma, Cancer Res. 58: 348-51, 1998.
14. Volpert, O., Jackson, D., Bouck, N., and Linzer, D. I. The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis, Endocrinology. 137: 3871-6, 1996.
15. Lin, S. B., Hsieh, S. H., Hsu, H. L., Lai, M. Y., Kan, L. S., and Au, L. C. Antisense oligodeoxynucleotides of IGF-II selectively inhibit growth of human hepatoma cells overproducing IGF-II, J Biochem (Tokyo). 122: 717-22, 1997.
16. Steller, M. A., Delgado, C. H., Bartels, C. J., Woodworth, C. D., and Zou, Z. Overexpression of the insulin-like growth factor-1 receptor and autocrine stimulation in human cervical cancer cells, Cancer Res. 56: 1761-5, 1996.
17. Steller, M. A., Delgado, C. H., and Zou, Z. Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells, Proc Natl Acad Sci U S A. 92: 11970-4, 1995.
18. Choy et al., xe2x80x9cMolecular mechanisms of drug resistance involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrationsxe2x80x9d Cancer Res. 48:2029-2035 (1988) 19. Fan et al., xe2x80x9cRibonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potentialxe2x80x9d Proc. Natl. Acad. Sci USA 93:14036-40 (1996)
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27. Hurta and Wright, xe2x80x9cMalignant transformation by H-ras results in aberrant regulation of ribonucleotide reductase gene expression by transforming growth factor-betaxe2x80x9d J. Cell Biochem 57:543-556 (1995)
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29. Nielsen et al.; Science (1991) 354:1497
30. Good and Nielsen; xe2x80x9cInhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNAxe2x80x9d, PNAS USA (1998) 95:2073-2076
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All of the above publications, patent applications and patents are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
Insulin-like growth factor II (IGF-II) is a 67 amino acid polypeptide growth factor that is widely expressed in the developing human embryonic tissues and is related to the growth and differentiation of various tissues. After birth, the expression is progressively extinguished in almost all human tissues. In adult humans, serum levels of approximately 100 ng/ml are mainly produced by the liver. The biological functions of IGF-II are mediated through its binding to either the IGF-II receptor (related to carbohydrate metabolism, motility of malignant cells and/or tumor-induced angiogenesis) or the IGF-I receptor (related to signal transduction pathway and mitogenesis).
IGF-II has been implicated in tumor progression and metastasis by a variety of mechanisms in many tumors (reviewed in (1, 2)). Tumors with extensive involvement of IGF-II include childhood tumors such as rhabdomyosarcoma, Wilms"" tumor and neuroblastoma. These tumors demonstrate overexpression of IGF-II, show existence of a paracrine or autocrine loop and result in inhibition of tumor growth or metastasis upon blockage of the loop. IGF-II contributes to tumor growth and metastasis to varying degrees in a variety of tumors including osteosarcoma, breast carcinoma, hepatoblastoma, germ cell tumors, hepatocellular carcinoma, adrenocortical carcinoma, lung tumors, leiomyosarcoma, brain tumors and colon carcinoma. Furthermore, the direct role of IGF-II in oncogenesis has been elucidated by transgenic mice and human cell lines overexpressing it (3-5) .
The human IGF-II gene is located on chromosome 11p15 just downstream of insulin gene and spans 30 kb (reviewed in (6) ;see FIG. 1). It consists of 9 exons of which exons 7, 8 and part of 9 encode a precursor protein. Exons 1, 4, 5, and 6 are each preceded by distinct promoters P1, P2, P3 and P4. Promoter P1 is active only in adult liver, while P2-4 are active in most fetal tissues. There are a few adult tissues that express low amount of transcripts from P2, 3 and 4 (fetal transcripts). Four major mRNA species (6 Kb, 4.8-5 Kb and 2.2 Kb for fetal transcripts and 5.3 Kb for adult transcript) have been identified which are generated from distinct promoters and by differential splicing. It appears that overexpression of IGF-II observed in various primary cancers and cell lines results from reactivation (in liver) or overexpression (in other organs) of fetal mRNA species whose expression is mainly derived from P3 and P4. These fetal transcripts contain unique 5xe2x80x2 untranslated regions (5xe2x80x2UTR containing exons 4 or 5 or 6) that are absent in the adult transcript derived from P1(5xe2x80x2UTR containing exons 1, 2 and 3).
Antisense oligonucleotides (AS-ODNs) have been widely utilized to inhibit gene expression in a target-specific manner by sequence-specific hybridization to target mRNA. In numerous studies, antisense oligonucleotide-mediated repression of oncogenes has revealed that these compounds are not only extremely useful for delineating biochemical mechanisms governing oncogenesis (7), but also considerably promising as novel therapeutic compounds for the treatment of human cancer (8, 9). In addition, relatively less toxicity has been attributed to oligonucleotide-based therapeutics (10).
A few studies (11, 15-17) have shown that certain antisense oligonucleotides targeted against human or mouse adult IGF-II transcripts were effective in interfering with tumor cell proliferation in vitro. In one study (15), the suppression of IGF-II production by an antisense oligonucleotide targeting the translation start site of human adult transcript has resulted in growth inhibition of human hepatocellular carcinoma cell lines, HuH-7 and HepG2. In another studies (16,17) utilizing human cervical cancer cell line, an antisense oligonucleotide targeting the protein coding region of IGF-II was shown to inhibit epidermal growth factor (EGF)-induced mitogenic effect.
Therefore, it would be desirable to identify antisense oligonucleotides directed against IGF-II which act to inhibit the expression and production of IGF-II with higher specificity and with less toxicity.
This invention is directed to antisense oligonucleotides which modulate the expression of the IGF-II genes and production of IGF-II in mammals and pharmaceutical compositions comprising such antisense oligonucleotides. This invention is also related to methods of using such antisense oligonucleotides for inhibiting tumor growth and metastasis in mammals.
Accordingly, in one of its composition aspects, this invention is directed to an antisense oligonucleotide, which oligonucleotide from about 3 to about 100 nucleotides comprising nucleotides complementary to the mammalian fetal IGF-II mRNA. The antisense oligonucleotide may be nuclease resistant and may have one or more phosphorothioate internucleotide linkages. The antisense oligonucleotide may further comprise additional nucleotides which are not complementary to the IGF-II mRNA. The oligonucleotides may comprise a sequence selected from group consisting of SEQ ID NOs:1 to 15 from Table 1.
This invention is also directed to an antisense oligonucleotide, which oligonucleotide from about 20 to about 100 nucleotides comprising nucleotides complementary to the mammalian adult IGF-II mRNA selected from the group consisting of SEQ ID NOs:17-31 from Table 2.
In another of its composition aspects, this invention is directed to a vector comprising an antisense oligonucleotide sequence from about 3 to 100 nucleotides comprising a sequence complementary to the 5xe2x80x2 untranslated region of mammalian fetal IGF-II mRNA.
In another of its composition aspects, this invention is directed to a vector comprising an antisense oligonucleotide sequence from about 20 to 100 nucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 17-31 in Table 2.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide from about 3 to about 100 nucleotides comprising nucleotides complementary to the mammalian fetal IGF-II mRNA. The oligonucleotides may comprise a sequence selected from group consisting of SEQ ID NOs:1 to 15 from Table 1.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide from about 20 to about 100 nucleotides comprising a sequence selected from the group consisting of SEQ ID NOs:17-31 from Table 2.
In one of its method aspects, this invention is directed to a method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides complementary to mammalian fetal IGF-l mRNA under conditions such that the growth of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent. The oligonucleotide may comprise a sequence selected from group consisting of SEQ ID NOs:1 to 15 from Table 1.
This invention is also directed to a method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide from about 20 nucleotides to about 100 nucleotides complementary to mammalian adult IGF-II mRNA selected from the group consisting of SEQ ID NOs:17-31 from Table 2 under conditions such that the growth of the tumor is inhibited.
In another of its method aspects, this invention is directed to a method for inhibiting the metastasis of a mammalian tumor comprising, administering to a mammal suspected of having a metastatic tumor an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides complementary to the mammalian fetal IGF-II mRNA under conditions such that the metastasis of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent. The oligonucleotides may comprise a sequence selected from group consisting of SEQ ID NOs:1 to 15 from Table 1.
This invention is also directed to a method for inhibiting the metastasis of a mammalian tumor comprising, administering to a mammal suspected of having a metastatic tumor an effective amount of an antisense oligonucleotide from about 20 nucleotides to about 100 nucleotides complementary to the mammalian adult IGF-II mRNA selected from the group consisting of SEQ ID NOs:17-31 from Table 2 under conditions such that the metastasis of the tumor is inhibited.